CN104568878A - Method for detecting copper ion based on graphene oxide chip - Google Patents
Method for detecting copper ion based on graphene oxide chip Download PDFInfo
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- CN104568878A CN104568878A CN201410832706.4A CN201410832706A CN104568878A CN 104568878 A CN104568878 A CN 104568878A CN 201410832706 A CN201410832706 A CN 201410832706A CN 104568878 A CN104568878 A CN 104568878A
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- graphene oxide
- slide
- dnazyme
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Abstract
The invention discloses a method for detecting copper ions based on a graphene oxide chip, and belongs to the field of biomedicines. The method for detecting the copper ions based on the graphene oxide chip mainly comprises the following steps: (1) preparing a slide with a graphene oxide dot matrix; (2) connecting a Cu<2+> correspondingly cut DNAzyme sequence with the slide with the graphene oxide dot matrix; (3) detecting the cutting action of Cu<2+> on the slide. By virtue of the principle that fluorescence of a graphene oxide can be quenched by AuNP, single chain DNAzyme containing Cu<2+> cut sites can be cut off after the action of Cu<2+> and is dissociated from single chain DNAzyme which is fixed on the graphene oxide and one end is modified by amino and the other end is marked by AuNP, the distance between AuNP and the surface of the graphene oxide is changed, and the fluorescence of the graphene oxide is changed, and with the combination of a high-flux micro array chip technique, Cu<2+> can be detected with low cost and high sensitivity.
Description
Technical field
The invention belongs to the heavy metal detection method in biomedical sector, especially a kind of based on graphene oxide biochip to Cu
2+detection method.
Background technology
Cu
2+in human body can and protein and various enzyme there is strong interaction, make them lose activity, also may enrichment in some organ of human body, if exceed the tolerant limit of human body, human body acute poisoning, subacute poisoning, slow poisoning etc. can be caused, people is known from experience and causes very large harm.Therefore, Cu
2+pollute the attention having caused people's height in medicine, food, environment etc.At present the research of copper ion detection method is got more and more, mainly comprise immunoassay, stripping voltammetry, test paper method etc., existing certain methods lacks higher sensitivity, also lower to the detection flux of sample, therefore setting up a kind of system that is highly sensitive, high-flux detection method is the research contents being badly in need of development at present.
Two scientist An Deliehaimu (Andre Geim) and the Constantine Nuo Woxiaoluofu (Konstantin Novoselov) of Univ Manchester UK in 2004 prepare Graphene first, and obtain the Nobel Prize in physics of 2010, receive the concern of whole world researchist.The Preparation of Graphene is simple, cost compare is low, there is more functional group on surface, as aldehyde radical, carboxyl etc., the fluorescence of the DNA energy cancellation graphene oxide self that golden nanometer particle (AuNP) marks, DNA enzymatic (DNAzyme) is a kind of DNA molecular with catalysis, has higher stability, when reacting with object, the change of structure can be caused (to work as Cu
2+during the DNAzyme of corresponding cutting, cause its DNAzyme structure to change, wherein a DNA chain is cut off, and departs from former matched sequence), thus be used to the detection of heavy metal.The DNAzyme of AuNP mark before cutting after, change with the distance on graphene oxide surface, the fluorescence intensity of graphene oxide changes, by the change of the fluorescence intensity of graphene oxide to Cu
2+concentration detects.Biochip is a kind of high-throughout instrument, utilizes the characteristic of graphene oxide, biochip is combined with graphene oxide, develops the Cu that a kind of sensitivity is higher
2+concentration detection method.
Summary of the invention
For Shortcomings in prior art, the invention provides a kind of in the Cu of graphene oxide chip
2+detection method, can low cost, high flux, high sensitivity is to Cu
2+detect.。
The present invention realizes above-mentioned technical purpose by following technological means.
Based on the detection method of graphene oxide chip to copper ion, comprise the steps:
(1) preparation has the slide of graphene oxide dot matrix:
1ml graphene oxide solution (0.3 mg/ml) point is at amido modified slide (long 76mm, wide 25mm, thick 1mm) on, after drying, with ultrapure water, on the slide with graphene oxide dot matrix, add 1 ml and to contain after the potpourri of 0.6 nM 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDCI) and 6.0 nM N-hydroxy thiosuccinimides (sulfo-NHS) at room temperature 12h.
(2) Cu is connected
2+the DNAzyme sequence of corresponding cutting is to the slide with graphene oxide dot matrix:
After the single stranded DNA zyme sequence that the amido modified other end AuNP in one end marks mixes according to the ratio of 1:1 with complementary one the DNAzyme sequence containing cleavage site, be placed on slide prepared by hybridizing box neutralization procedure (1) and carry out chemical reaction 12h, wash away unnecessary probe with ultrapure water; The fluorescence intensity F of graphene oxide on scanning record slide
2.
(3) Cu
2+detect:
Joined by the copper ion solution of variable concentrations on the slide processed through step (2), reaction below 15min, with ultrapure water, scanning record graphene oxide is at Cu
2+fluorescence intensity F after cutting action
3.
Mechanism of the present invention is: the Cu adding variable concentrations
2+by the effect to DNAzyme in step (2) under different time, Cu
2+cutting be connected to graphene oxide have AuNP mark DNAzyme after, the structure of DNAzyme changes: have the single stranded DNA zyme of cleavage site to depart from graphene oxide surface, the distance being fixed on single stranded DNA zyme on graphene oxide and graphene oxide surface containing AuNP mark with cut before change, the fluorescence intensity of graphene oxide is caused to change, with scanning after ultrapure water and analysis, by graphene oxide fluorescence intensity Cu
2+change before and after effect, works as Cu
2+cutting action rear oxidation Graphene fluorescence intensity F
3/ Cu
2+the fluorescence intensity F of graphene oxide before cutting action
2ratio close to 0 time, illustrate in solution to there is Cu
2+, detection sensitivity during the method detection copper ion can reach 0.1nM.
The invention has the beneficial effects as follows:
(1) the present invention combines graphene oxide and microarray chip technology, and energy low cost, Gao Tong Liang ﹑ high sensitivity are to Cu
2+concentration detects.
(2) the present invention fully use AuNP to mark DNAzyme sequence energy cancellation biochip on fluorescence on graphene oxide, but when DNAzyme at Cu
2+under effect, structure changes, and AuNP is different from the distance of graphene oxide, the principle that the efficiency of its cancellation is different, to Cu
2+detect, relatively simple and effective.
Accompanying drawing explanation
Fig. 1 is schematic flow sheet of the present invention.
Fig. 2 is Cu
2+cutting DNA zyme course of reaction figure.
Embodiment
Below in conjunction with accompanying drawing and specific embodiment, the present invention is further illustrated, but protection scope of the present invention is not limited to this.
1. the preparation of amido modified slide: add 5 ml hydrogen peroxide (H in the beaker being placed with slide
2o
2), then add the 15 ml concentrated sulphuric acid (H with transfer pipet
2sO
4), slowly vibration or leave standstill 30 min and make it abundant reaction on shaking table, this reaction can make surface hydroxylation, outwells the liquid of step reaction, by washed with de-ionized water 3 times.First use washes of absolute alcohol 2 times, then pour 20 ml absolute ethyl alcohols into, the hydroxylation silicon chip of reacted acquisition is transferred in beaker, with washes of absolute alcohol 3 times.Outwell ethanol after having cleaned, add rapidly the mixed liquor (volume ratio is 1:15) of APTES (APTES) and absolute ethyl alcohol, or first add 15ml absolute ethyl alcohol, then add 1 ml APTES with transfer pipet, on shaking table, 2 h are reacted in jolting.
2. the preparation of graphene oxide: according to the Hummer method improved, in there-necked flask, add 3g crystalline graphite powder, 1.5 g NaNO
3with put into thermostat water bath after the 69 ml concentrated sulphuric acids and stir.1g KMnO is added after reaction 1h
4, react at 35 DEG C after 5 hours and add 150 ml deionized waters.React 30 min at temperature 98 DEG C after, then add deionized water, the 5 ml H of 50 ml
2o
2and 250 10% watery hydrochloric acid of ml, solution is poured in the large beaker of 1000 ml, wash to pH value be 5-6.
3. preparation has the slide of graphene oxide dot matrix: 1 ml graphene oxide solution (0.3 mg/ml) point, on amido modified slide (long 76mm, wide 25mm, thick 1mm), after drying, uses ultrapure water.Add 1 ml EDCI(0.6 nM) and sulfo-NHS(6.0 nM) at room temperature 12h after potpourri.
4. graphene oxide activated for 0.7ul and the concentration of modifying through terminal amino group are 5pmol Cu by linking objective DNAzyme sequence: synthetic DNA zyme sequence on graphene oxide chip
2+the DNAzyme potpourri of corresponding cutting connects, and obtains the chip connecting DNAzyme.The DNA sequence dna used is as follows: a. 5 ￠-TTCTTT CTAA TACG GC TTACC-3 ￠, b.5 ￠-NH2-GGTAAGCCTGGGCCTCTTTC TTT TTA AG AAAGAAC-3 the ￠ marked with AuNP is held with 3 ', the DNA of double-strand pouch-type conformation is formed, Cu by a and b sex change
2+can the specific site of specific this DNA sequence dna of cutting, wherein sequence a 17-24 position and sequence b 1-8 bit base complementation from 5 ￠ hold from 5 ￠ ends, and sequence b 15-22 position and sequence b 25-32 bit base complementation from 5 ￠ hold from 5 ￠ ends, 2h is hybridized after mixing under 37 degree according to the ratio of 1:1, liquid after 0.7ul hybridization drips 1) surface of glass slide prepared, chemical reaction 12 h is carried out in hybridizing box, and be fixed on graphene oxide dot matrix, the AuNP that DNA sequence dna marks is by the fluorescence intensity of fluorescent energy transmission energy cancellation graphene oxide, at 532nm, chip is scanned, (F after the fluorescence intensity cancellation of graphene oxide
2) with cancellation before (F
1) ratio be 80%-95%.
5. Cu
2+detection: add 0.1 nM Cu
2+solution (sodium ascorbates containing 50 uM) carries out cutting 10-15 min to being connected to DNAzyme on graphene oxide, after cutting action, the structure of DNAzyme changes, the DNAzyme of AuNP mark and the distance on graphene oxide surface change, at 532nm, chip is scanned, the fluorescence intensity can observing graphene oxide occurs further to change, its fluorescence intensity (F
3) and Cu
2+graphene oxide fluorescence intensity (F before cutting
2) ratio be close to 0, under same experimental conditions, carry out repeated experiment, above-mentioned experimental phenomena is consistent, show chip surface fluorescence intensity change come from be connected to graphene oxide surface DNAzyme sequence at Cu
2+the change of the lower structure of effect, the sensitivity of detection can reach 0.1 nM.The higher detection sensitivity reported is 1 nM.(Y. Hao, L. Liu, Y. Long, J. Wang, Y. Liu, F. Zhou, Biosens. Bioelectron., 2013,41,723 – 729.) improve 10 times in sensitivity.
The present invention has carried out control experiment under identical treatment conditions, by other heavy metal ion of same concentrations as Pb
2+, Mg
2+, Al
3+deng cutting, its fluorescence intensity does not have significant change.The above results shows, Cu
2+can effectively cut DNAzyme, by the impact on graphene oxide fluorescence intensity, thus to Cu
2+concentration carries out specific detection.
The present invention has carried out control experiment under identical treatment conditions, by other heavy metal ion of same concentrations as Pb
2+, Mg
2+, Al
3+deng cutting, its fluorescence intensity does not have significant change.The above results shows, Cu
2+can effectively cut DNAzyme, by the impact on graphene oxide fluorescence intensity, thus to Cu
2+concentration carries out specific detection.
Described embodiment is preferred embodiment of the present invention; but the present invention is not limited to above-mentioned embodiment; when not deviating from flesh and blood of the present invention, any apparent improvement that those skilled in the art can make, replacement or modification all belong to protection scope of the present invention.
Claims (5)
1., based on the detection method of graphene oxide chip to copper ion, it is characterized in that:
(1) preparation has the slide of graphene oxide dot matrix;
(2) to the DNAzyme of the amido modified other end AuNP mark in one end and containing Cu
2+after cleavage site single stranded DNA zyme sex change hybridization, be connected to there is graphene oxide dot matrix slide on, the fluorescence intensity F of graphene oxide on scanning record slide
2;
(3) joined by the copper ion solution of variable concentrations on the slide processed through step (2), reaction below 15min, with ultrapure water, scanning record graphene oxide is at Cu
2+fluorescence intensity F after cutting action
3.
2. detection method according to claim 1, it is characterized in that: described step (1) be by 1ml graphene oxide solution point on amido modified slide, after drying, with ultrapure water, then to add after 1ml EDCI and sulfo-NHS potpourri at room temperature 12h.
3. detection method according to claim 2, is characterized in that: the concentration of described graphene oxide solution is 0.3 mg/ml; The concentration of EDCI solution is 0.6 nM; The concentration of sulfo-NHS solution is 6.0 nM.
4. detection method according to claim 1, is characterized in that: described step (2), and the amido modified other end in one end has AuNP marker DNA zyme and contains Cu
2+after an other DNAzyme sequence of cleavage site according to the ratio of 1:1 in hybridizing box and 1) slide prepared carries out chemical reaction 12h, wash away unnecessary DNA sequence dna with ultrapure water, carry out scanning analysis.
5. detection method according to claim 1, is characterized in that: work as Cu
2+the fluorescence intensity F of cutting action rear oxidation Graphene
3/ Cu
2+graphene oxide fluorescence intensity F before cutting action
2ratio close to 0 time, illustrate in solution to there is Cu
2+, detection sensitivity during the method detection copper ion can reach 0.1nM.
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Cited By (3)
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| CN105842209A (en) * | 2016-03-18 | 2016-08-10 | 中国科学院合肥物质科学研究院 | Paper sensor for rapid on-site detection of fluorine ions in water and preparation method thereof |
| CN109557155A (en) * | 2019-01-17 | 2019-04-02 | 杭州电子科技大学 | It is a kind of based on graphene-In Glassy Carbon Electrode Modified With Nano-gold preparation method and application |
| CN111965151A (en) * | 2020-07-31 | 2020-11-20 | 江苏大学 | Graphene oxide biochip and preparation method and application thereof |
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2014
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| US20110123982A1 (en) * | 2007-07-16 | 2011-05-26 | Yi Lu | Nucleic acid based fluorescent sensor for copper detection |
| US20120149119A1 (en) * | 2007-08-10 | 2012-06-14 | Yi Lu | Nucleic acid based fluorescent sensor for mercury detection |
| CN103031375A (en) * | 2012-12-10 | 2013-04-10 | 江苏大学 | DNA (deoxyribonucleic acid) methylation detection kit and detection method |
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Cited By (3)
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| CN105842209A (en) * | 2016-03-18 | 2016-08-10 | 中国科学院合肥物质科学研究院 | Paper sensor for rapid on-site detection of fluorine ions in water and preparation method thereof |
| CN109557155A (en) * | 2019-01-17 | 2019-04-02 | 杭州电子科技大学 | It is a kind of based on graphene-In Glassy Carbon Electrode Modified With Nano-gold preparation method and application |
| CN111965151A (en) * | 2020-07-31 | 2020-11-20 | 江苏大学 | Graphene oxide biochip and preparation method and application thereof |
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